The present invention relates to eukaryotic cells which express at least one HLA-G isoform on their surface and to their uses, in particular for obtaining a medicament for modulating the cytolytic activity of NK cells in pathologies in which these NK cells are activated or inhibited and as a model system for use, in particular, in a method for screening antineoplastic substances.
The present invention also relates to transgenic animals which specifically express at least one HLA-G isoform.
The antigens of the major histocompatibility complex (MHC) divide into several classes, i.e. class I antigens (HLA-A, HLA-B and HLA-C), which exhibit 3 globular domains (xcex11, xcex12 and xcex13) and whose xcex13 domain is associated with xcex22 microglobulin, the class II antigens (HLA-DP, HLA-DQ and HLA-DR) and the class III antigens (complement).
In addition to the abovementioned antigens, the class I antigens include other antigens known as non-classical class I antigens, in particular the HLA-E, HLA-F and HLA-G antigens; this latter antigen, in particular, is expressed by the extravillous trophoblasts of the normal human placenta.
The sequence of the HLA-G gene (HLA-6.0 gene) has been described by Geraghty et al., (Proc. Natl. Acad. Sci. USA, 1987, 84, 9145-9149): it comprises 4396 base pairs and exhibits an intron/exon organization which is homologous with that of the HLA-A, -B and -C genes. More precisely, this gene comprises 8 exons, 7 introns and an untranslated 3xe2x80x2 end; the 8 exons correspond, respectively, to: exon 1: signal sequence, exon 2: xcex11 extracellular domain, exon 3: xcex12 extracellular domain, exon 4: xcex13 extracellular domain, exon 5: transmembrane region, exon 6: cytoplasmic domain I, exon 7: cytoplasmic domain II (untranslated), exon 8: cytoplasmic domain III (untranslated) and untranslated 3xe2x80x2 region (Geraghty et al., loc. cit.; Ellis et al., J. Immunol., 1990, 144, 731-735; Kirszenbaum M. et al., Oncogeny of hematopoiesis. Aplastic anemia Eds. E. Gluckman, L. Coulombel, Inserm Symposium/John Libbey Eurotext Ltd). However, the HLA-G gene differs from the other class I genes in that the in-frame translation termination codon is located in the second codon of exon 6; as a consequence, the cytoplasmic region of the protein encoded by this HLA-6.0 gene is considerably shorter than that of the cytoplasmic regions of the HLA-A, -B and -C proteins.
These HLA-G antigens are mainly expressed by the cytotrophoblastic cells of the placenta and are regarded as playing a role in the protection of the foetus (no rejection by the mother). Furthermore, to the extent that the HLA-G gene is monomorphic, it may also be involved in the growth or function of the placental cells (Kovats et al., Science, 1990, 248, 220-223).
Other studies dealing with this non-classical class I antigen (Ishitani et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 3947-3951) have shown that the primary transcript of the HLA-G gene can be spliced in several ways and produces at least 3 distinct mature mRNAs: the primary transcript of HLA-G gives one complete copy of 1200 bp (G1), one fragment of 900 bp (G2) and one fragment of 600 bp (G3).
The G1 transcript does not contain exon 7 and corresponds to the sequence described by Ellis et al. (loc. cit.), i.e. it encodes a protein which comprises a leader sequence, three external domains, a transmembrane region and a cytoplasmic sequence. The G2 mRNA does not contain exon 3, i.e. it encodes a protein in which the xcex11 and xcex13 domains are linked directly; the G3 mRNA contains neither exon 3 nor exon 4, i.e. it encodes a protein in which the xcex11 domain and the transmembrane sequence are linked directly.
The splicing which prevails for obtaining the HLA-G2 antigen entails an adenine (A) (originating from the domain encoding xcex11) being linked to an AC sequence (derived from the domain encoding xcex13), leading to the creation of an AAC (asparagine) codon in place of the GAC (aspartic acid) codon which is encountered at the beginning of the sequence which encodes the xcex13 domain in HLA-G1.
The splicing which is generated for obtaining HLA-G3 does not entail the formation of a new codon in the splicing zone.
The authors of this article have also analysed the different proteins which are expressed: the 3 mRNAs are translated into protein in the 0.221-G cell line.
The authors of this article conclude that HLA-G plays a fundamental role in protecting the foetus with regard to a maternal immune response (induction of immune tolerance). However, it is pointed out that the role of the G3 protein, which does not contain the xcex13 domain, is not established.
Some of the inventors have recently demonstrated the existence of other spliced forms of HLA-G mRNA: i.e. the HLA-G4 transcript, which does not include exon 4; the HLA-G5 transcript, which includes intron 4 between exons 4 and 5, thereby giving rise to a change in the reading frame during translation of this transcript, in particular to the appearance of a stop codon after amino acid 21 of intron 4; and the HLA-G6 transcript, which possesses intron 4 but which has lost exon 3 (Kirszenbaum M. et al., Proc. Natl. Acad. Sci. USA, 1994, 91, 4209-4213; European Application EP 0 677 582; Kirszenbaum M. et al., Human Immunol., 1995, 43, 237-241; Moreau P. et al., Human Immunol., 1995, 43, 231-236); they have also demonstrated that these different transcripts are expressed in several types of human foetal and adult cells, in particular in lymphocytes (Kirszenbaum M. et al., Human Immunol., 1995, loc. cit.; Moreau P. et al., Human Immunol., 1995, loc. cit.).
There are therefore at least 5 different HLA-G mRNAs which potentially encode 5 isoforms of HLA-G.
Although the foetus can be regarded as being a semiallograft, the foetal cells survive and are not rejected by the mother; it has emerged that the HLA-G molecules which are expressed on the surface of the trophoblasts protect the foetal cells from lysis by the maternal natural killer. (NK) cells (Carosella E. D. et al., C.R. Acad. Sci., 318, 827-830; Carosella E. D. et al., Immunol. Today, 1996, 407-409).
Earlier studies have demonstrated that expression of HLA-G molecules on the surface of target cells protects the said target cells from the lytic activity of the NK cells of the decidual layer of the maternal endometrium (Chumbley G. et al., Cell Immunol., 1994, 155, 312-322; Deniz G. et al., J. Immunol., 1994, 152, 4255-4261). It is to be noted that these target cells are obtained by means of transfection with vectors which contain the HLA-G genomic DNA, which is potentially able to generate all the alternative transcripts.
The NK cells express receptors for MHC class I molecules (killer inhibitory receptors or KIR or NKIR for NK inhibitory receptors), which receptors are responsible for inhibiting cytotoxicity when these HLA molecules, acting as ligands, are recognized by these receptors; for example, Pazmany L. et al., (Science, 1996, 274, 792-795) showed that the expression of HLA-G protected LCL 721.221 (B lymphoma cell line) target cells, which were transfected with the HLA-G gene, from lysis. These cells are ordinarily sensitive to NK cells; they furthermore identified the receptors on the NK cells which recognize HLA-G, namely the NKIR1 and NKIR2 receptors, which belong to the immunoglobulin (p58) superfamily and which are able to distinguish between two dimorphic groups of HLA-C molecules; HLA-G could be the natural ligand of the NK cell receptors; thus, some of the inventors have shown that NK cells do not express any HLA-G transcript; this result confirms that the expression products of the HLA-G gene probably play a role in immunotolerance (Teyssier M. et al., Nat. Immunol., 1995, 14, 262-270).
In view of the important role which the HLA-G molecule may play both in pathologies in which the NK cells are particularly active (autoimmune diseases, transplantations) or in which they are, on the other hand, inhibited (abnormal presence of HLA-G molecules, in particular on certain tumours or in viral infections), the inventors surprisingly found, in continuing their studies, that it was possible to express one single isoform on the cell surface and to regulate its quantitative and qualitative expression and therefore to control its use.
The present invention relates to eukaryotic cells which are obtained by genetic modification, characterized in that they are transfected with an expression vector which contains at least one cDNA encoding an HLA-G isoform which is selected from the group consisting of the HLA-G2, HLA-G3, HLA-G4, HLA-G5 and HLA-G6 isoforms.
The present invention also relates to eukaryotic cells which are obtained by genetic modification, characterized in that they are transfected with an expression vector which contains a suitable origin of replication, a selection marker such as a gene for resistance to an antibiotic, the RSV viral promoter and a cDNA which encodes an HLA-G isoform which is selected from the group consisting of the HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5 and HLA-G6 isoforms.
These cells efficiently express the different isoforms in a glycosylated form which is similar to that encountered under biological conditions.
According to one advantageous embodiment of the said transfected eukaryotic cells, the said cDNA encodes an isoform which contains at least one extracellular domain, in particular the xcex11 domain.
Surprisingly, the cytolytic activity of the NK cells is inhibited in the presence of the said eukaryotic cells which have been transfected in this way irrespective of the isoform expressed (membrane isoform or secreted isoform).
The said eukaryotic cells may be derived from any animal and may in particular be mammalian cells, more especially human cells.
Both the transfected cells according to the invention and eukaryotic cells which are transfected with a vector expressing the HLA-G1 isoform can be used:
in order to obtain an immunomodulatory medicament for inhibiting the activity of killer cells, in particular NK cells and/or for inhibiting the primary allogeneic response; an immunomodulatory product which has a general action or an immunomodulatory product which has a specific action, and which is in particular suitable for protecting a transplanted organ, is obtained depending on the type of eukaryotic cells transfected; transfected haematopoietic stem cells, hepatic cells and renal cells may be mentioned as non-limiting examples; unexpectedly, the said eukaryotic cells protect the target cells from attack by all classes of NK cells irrespective of the HLA-G isoform expressed; these eukaryotic cells are particularly suitable for use in the prevention of graft rejection (allografts and xenografts), in the prevention of repeat abortions and in the treatment of autoimmune diseases and pathologies or situations in which killer cells are activated in a general manner;
in order to obtain a medicament for relieving the inhibitory function of the isoform(s) expressed by the said cells with regard to killer cells, in particular NK cells, in pathologies in which these killer cells are inhibited by the HLA-G class I major histocompatibility complex molecule; thus, solid tumours express some HLA-G isoforms; this expression protects these cancerous cells from the lysis which is induced by NK cells;
in order to produce antineoplastic vaccines: production of antibodies which block the HLA-G antigen and thereby reinduce the activity of the NK cells;
in order to produce human cells which are capable of being transplanted into a specific organ and of protecting this latter organ from the lysis which is induced by killer cells, in particular NK cells;
as a model for studying the interaction of HLA-G and immunocompetent cells, in particular killer cells, more especially NK cells, in order to find effectors which are capable of modulating the antineoplastic response and inhibiting the HLA-G expression which is induced in some cancers while at the same retaining the expression of the classic HLA antigens; this enables them to be used for obtaining a medicament for relieving the inhibitory function of the isoform(s) expressed by the said cells with regard to killer cells, in particular NK cells, in pathologies, such as cancers, in which these NK cells are inhibited by the HLA-G class I major histocompatibility complex molecule;
in order to produce non-human transgenic mammals which express a specific HLA-G isoform and which are able to produce tissues which express the said isoform (xenograft donors) and/or which are able to form models for studying the regulation and the function of the different tolerance-linked HLA-G isoforms in autoimmune diseases and during transplanting.
In order to produce the said transgenic animals, in particular transgenic mice, at least one copy of a segment containing a cDNA which encodes an HLA-G isoform linked to a suitable promoter is introduced into the cells of a mouse embryo at an early stage.
The present invention also relates to a purified and isolated receptor, characterized in that the receptor is capable of binding at least one HLA-G isoform which is expressed on the surface of a eukaryotic target cell such as defined above, in that the said receptor is expressed on the surface of T cell lines which neither express any CD94, KIR1 or KIR2 inhibitory receptor nor at least some membrane receptors which are specific for T cells, and in that the formation of the HLA-G isoform/receptor complex inhibits the lysis of the target cells by the said T cell lines.
Advantageously, the said T cell lines do not express at least the CD3 and xcex1xcex2 receptors or do not express any membrane receptor which is specific for T cells.
The immature leukaemic T cell line designated YT2C2 is a cell line of this type since it does not express any CD94, KIR1 or KIR2 inhibitory receptor nor receptors which are specific for T cells, such as the CD3 and xcex1xcex2 receptors.
In order to isolate the said receptor, it is possible:
a) either to incubate the said T cell lines with an HLA-G isoform, in particular with the soluble HLA-G5 isoform, isolate the resulting HLA-G/receptor complex by treating cell lines with trypsin, dissociate the receptor from the HLA-G molecule by treating with a denaturing agent such as a detergent, and separate the receptor, either by passing the resulting mixture through an immunoaffinity column coupled to an anti-HLA-G antibody and recovering the free receptor in the eluate, or by subjecting the said mixture to a suitable electrophoresis.
b) or to clone the said receptor in accordance with the method described in Colonna M. et al., Science, 1995, 268, 405-408.
The present invention also relates to a process for studying the binding affinity of an HLA-G isoform for a KIR receptor or a receptor such as defined above (receptor having NK activity), characterized in that the process comprises:
transfecting a eukaryotic host cell with an expression vector containing a cDNA encoding an HLA-G isoform,
culturing the said host cells such that they express the said HLA-G isoform on their surface,
bringing the said transfected eukaryotic cells into contact with killer cells, such as the NK cells or the T cell lines as defined above, in the presence of substances which activate or inhibit the HLA-G and/or substances which activate or inhibit the KIR receptor or the receptor having NK activity in accordance with the invention and
measuring the quantity of HLA-G isoform/receptor complex.
The present invention also relates to products which comprise a eukaryotic cell expressing at least one HLA-G isoform as defined above and a factor for stimulating the expression of HLA-Gs, as combination products for simultaneous or separate use or use which is staggered over time, in the prevention or treatment of pathologies or situations in which the killer cells are activated, such as transplants, repeat abortions or autoimmune diseases.
In accordance with the invention, the said stimulatory factor is selected from the group consisting of corticoids and cytokines.